C-type lectin receptors differentially induce th17 cells and vaccine immunity to the endemic mycosis of North America

Abstract

Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

Fig. 1. Card9 is required for vaccine control and the acquisition of adaptive immunity to dimorphic fungi

(A) Wild type and Card9^−/− mice were subcutaneously vaccinated with 10⁵ or 10⁶ live B. dermatitidis (B.d.) yeast and survival recorded when mice were moribund. N=10 mice/group; representative of 2 experiments. (B) Mice were vaccinated with 10⁶ heat-killed (HK) B.d., 10⁷ HK or live H. capsulatum (H.c.) yeast, or 10⁶ FKS of C. posadasii (C.p.) or not as a control and challenged with 2 × 10³ B.d or 2 × 10⁵ H.c. yeast two weeks or 80 C.p. spores three weeks after booster vaccination. Lung CFU were determined when unvaccinated mice were moribund ≥ 14 days post-infection with B.d. or C.p. and at day 13 post-infection with H.c.. Data are the mean ± SEM (n=8–12 mice/group); representative of 3-5 experiments. * P < 0.05 vs. vaccine-induced reduction in lung CFU in wild type mice. Numbers indicate the n-fold difference in lung CFU vs. unvaccinated controls. (C) Wild type and Card9^−/− mice received 10⁶ naïve, CD4⁺ purified 1807 T cells and were vaccinated with HK B.d. or H.c. yeast, FKS from C.p. or not. Five weeks later, mice were challenged with 2 × 10⁴ B.d., 2 × 10⁵ H.c. yeast or 10⁶ FKS from C.p. and the number of cytokine producing 1807 cells enumerated by FACS at day 4 post-infection. Data are the mean ± SEM of 3 independent experiments (n=16–20 mice/group). * P < 0.05 vs. vaccinated wild type controls. (D) The frequency of cytokine producing 1807 cells in the lung at day 4 post-infection. Data are the mean ± SEM (n=16–20 mice/group); representative of 3 experiments. * P < 0.05 vs. vaccinated wild type controls. ND; not detected.

Fig. 2. Card9 is dispensable for T cell expansion and activation

(A) Wild type and Card9^−/− mice received an adoptive transfer of 10⁶ CD4⁺ purified, CFSE-labeled, naïve 1807 Tg cells and were vaccinated with 10⁶ HK B.d. yeast or not. Transferred 1807 cells were harvested from the sdLN and proliferation was assessed by enumerating the frequency of CFSE^low cells (mean ± SEM (n=4–6 mice/group); representative of 3 experiments) at day 7 post-vaccination. (B) At serial time points post-vaccination, the frequency of activated (CD44^hi) 1807 Tg cells was determined in the sdLN and in the lung at day 4 post-infection. Data are the mean ± SEM (n=4–6 mice/group); representative of 5 experiments. (C) The number of activated (CD44^hi) 1807 Tg cells for (B). Data are the mean ± SEM (n=4–6 mice/group); representative of 5 experiments. * P < 0.05 vs. vaccinated wild type controls.

Fig. 3. Helper T cells fail to differentiate without Card9 signaling

(A) Primed T cells from sdLN of wild type and Card9^−/− mice were co-cultured ex vivo with CW/M extract for three days and cytokine production measured by ELISA. Data are the mean ± SEM (n=6 mice/group); representative of 3 experiments. * P < 0.05 vs. vaccinated wild type controls. (B) Bone marrow derived dendritic cells (BMDCs) from wild type and Card9^−/− mice were co-cultured with B.d. vaccine yeast and CD4⁺ purified naïve 1807 cells for three days, and cytokine production was measured by ELISA. Data are the mean ± SEM (n=3–5 mice/group); representative of 5 experiments. * P < 0.05 vs. vaccinated wild type controls. (C) CD4⁺ purified 10⁶ naïve wild type 1807 Tg cells were adoptively transferred into wild type and Card9^−/− mice prior to vaccination with 10⁷ HK B.d. yeast. Effector T cells were harvested from the subcutaneous tissue 10 days post-vaccination and the frequency of cytokine producing cells was determined by FACS analysis. Data are the mean ± SEM (n=6 mice/group); representative of 3 experiments. * P < 0.05 vs. vaccinated wild type controls. (D) The changes in cytokine transcript and protein were measured by real-time RT-PCR and ELISA for BMDCs cultured with vs. without yeast. Data are the mean ± SEM (n=13–15 mice/group). * P < 0.05 vs. wild type controls.

Fig. 4. Soluble Dectin-1-Fc, Dectin-2-Fc, and Mincle-Fc proteins bind three dimorphic fungi

Fungal cells were incubated with Fc fragment alone or Fc fusion proteins as indicated by different colors, followed by staining with phycoerythrin (PE)-conjugated anti-human Fc antibody, and analyzed by FACS (A) and microscopy (B). As a negative control, antibody-stained fungal cells without any Fc proteins are indicated by grey filled histograms in (A). Scale bar, 10 μm. Data are representative of two independent experiments.

Fig. 5. Differential recognition of three dimorphic fungi by surface bound Dectin-1, Dectin-2, and Mincle receptors

(A) BWZ cells and a subline expressing Dectin-1-CD3ζ (Dectin-1), as well as B3Z cells expressing Dectin-2, Mincle, FcRγ chain, Dectin-2 + FcRγ, Dectin-2 + FcRγm (a signaling-defective FcRγ chain), Mincle + FcRγ, or Mincle + FcRγm were stimulated with the indicated numbers of heat-killed B.d. (left column), H.c. (middle column), or C.p. (right column). After 18 hours, lacZ activity was measured using a colorimetric assay and expressed as OD 560/620 values. Data are the mean ± SD of duplicate wells. (B) Reporter cells expressing Dectin-1-CD3ζ, Dectin-2 + FcRγ, or Mincle + FcRγ were pre-stained with CFSE and incubated for 6 hours with heat killed fungal cells pre-stained with LIVE/DEAD Violet staining. The numbers indicate the frequencies of B3Z/BWZ cells bound by fungi as analyzed by FACS. Data are representative of two independent experiments.

Fig. 6. FcRγ and Dectin-2 are required for T-cell differentiation and vaccine immunity to B. dermatitidis infection

(A) At day 7 post-vaccination cells harvested from the sdLN of vaccinated wild type, Dectin-2^−/−, FcRγ^−/−, and Mincle^−/− mice were co-cultured ex vivo with CW/M extract and cytokine production measured by ELISA. Data are the mean ± SEM (n=6 mice/group); representative of 3 experiments. * P < 0.05 vs. wild type controls. (B) Wild type, Dectin-2^−/−, FcRγ^−/−, and Mincle^−/− BMDCs were co-cultured with B.d. vaccine yeast and CD4⁺ purified, naïve 1807 cells for 3 days, and cytokine production measured by ELISA. Data are the mean ± SEM (n=3–5 mice/group); representative of 3 experiments. * P < 0.05 vs. wild type controls. (C) Wild type, Dectin-2^−/−, FcRγ^−/−, and Mincle^−/− mice received 10⁶ naïve, CD4⁺ purified, wild type 1807 T-cells and were vaccinated with 10⁷ HK B.d. yeast. 10 days post-vaccination, the frequency of cytokine producing 1807 T cells harvested from the site of vaccination was determined by FACS analysis. Data are the mean ± SEM (n=6 mice/group); representative of 3 experiments. * P < 0.05 vs. wild type controls. (D) Mice described in (C) were challenged with 2 × 10⁴ strain 26199 yeast 5 weeks post-vaccination and the numbers and frequencies of cytokine producing 1807 cells were enumerated by FACS at day 4 post-infection. Data are the mean ± SEM (n=4–6 mice/group); representative of 4 experiments. * P < 0.05 vs. vaccinated wild type controls. (E) Wild type, Dectin-2^−/−, FcRγ^−/−, Mincle^−/−, and chimeric Syk^+/+ or Syk^−/− mice were vaccinated with 10⁶ HK B.d. yeast or not as a control and challenged with 2 × 10³ strain 26199 yeast two weeks after booster vaccination. Lung CFU were determined when unvaccinated mice were moribund (>14 days post-infection). Data are the mean ± SEM (n=8–12 mice/group); representative of 4 experiments. Numbers indicate the n-fold change in lung CFU vs. unvaccinated controls. * P < 0.05 vs. vaccine-induced reduction in lung CFU in wild type or Syk^+/+ mice.

Fig. 7. FcRγ and Dectin-2 are required for T-cell differentiation and adaptive immunity to C. posadasii and H. capsulatum

(A) 10⁶ naïve CD4⁺ purified, wild type 1807 Tg cells were transferred into wild type, Dectin-2^−/−, FcRγ^−/− and Mincle^−/− mice prior to vaccination with 10⁶ FKS of C.p.. Two weeks after the boost, the mice were challenged with 10⁶ FKS. At day 4 post-infection, the numbers and frequencies of cytokine producing 1807 cells in vaccinated mice were determined by FACS analysis. Data are the mean ± SEM (n=4–6 mice/group); representative of 2 experiments. * P < 0.05 vs. vaccinated wild type controls. (B) Wild type, Dectin-2^−/−, FcRγ^−/− and Mincle^−/− mice were vaccinated with 10⁶ FKS or 5 × 10⁴ viable spores of the ΔT strain (or not as a control) and challenged with 50-80 C. posadasii spores four weeks later. Lung and spleen CFU were determined 14 days post-infection. Data are the averages ± SEM of two independent experiments (n=8–12 mice/group/experiment); representative of 2 experiments. Numbers indicate the n-fold change in lung CFU vs. unvaccinated controls. * P < 0.05 vs. vaccine-induced reduction in lung CFU in wild type mice. (C) Same as (A), but mice were vaccinated with 10⁷ HK H.c. G217B yeast and challenged with 10⁵ H.c. yeast. Data are the mean ± SEM (n=4–6 mice/group); representative of 2 experiments. * P < 0.05 vs. vaccinated wild type controls. (D) Wild type, Dectin-2^−/−, FcRγ^−/− and Mincle^−/− mice were vaccinated with 10⁷ live or HK H.c. G217B yeast (or not as a control) and challenged with 10⁵ H.c. yeast two weeks after booster vaccination. Lung CFU were determined 13 days post-infection. Data are the mean ± SEM (n=8–12 mice/group); representative of 2 experiments. Numbers indicate the n-fold change in lung CFU vs. unvaccinated controls. * P < 0.05 vs. vaccine-induced reduction in CFU in wild type mice.